Method for preparing polybiotinylated compounds

ABSTRACT

The present invention relates to a novel method for preparing compounds having the formula (I), 
     
       
         
         
             
             
         
       
     
     where
         X is biotin or       

     
       
         
         
             
             
         
       
     
     Y being biotin or 
     
       
         
         
             
             
         
       
     
     Z being biotin or 
     
       
         
         
             
             
         
       
     
     V being biotin or 
     
       
         
         
             
             
         
       
     
     It also relates to compounds having the formula (I) and their use in clinical and industrial diagnosis.

The present invention relates to the field of diagnosis. In particular,it relates to a method for producing polybiotinylated dendrimercompounds useful for signal amplification, in particular during thedetection of an analyte in a diagnostic test.

Probes which allow the detection of analytes such as proteins andnucleic acids are widely used and are tools of choice for in vitro andin vivo diagnosis. However, it may be difficult to detect these analytesbecause they are not always present in sufficiently large quantities. Adetection amplification system is therefore necessary.

Several techniques have been developed for detecting analytes ininsufficient quantities. These techniques involve either increasing thequantity of analyte, for example with PCR or NASBA techniques, oramplifying the detection, for example by using structures havingmultiple markers.

Thus, patent application EP0774119A describes conjugates having multiplemarkers comprising a polymer support having a maximum of 100 monomerunits, which contains 1 to 10 molecules of haptene and 1 to 10 markergroups, and also the preparation of these conjugates by solid-phasesynthesis (a) by introducing, at predetermined positions of the support,monomer derivatives which are coupled covalently to haptene moleculesand/or marker groups and/or (b) by coupling haptene molecules and/ormarker groups to reactive groups of the support, after the synthesis.

Dendrimers are clearly defined macromolecules which have multiplebranches and which terminate in multiple peripheral functional groups.One of the advantages of dendrimers, due to the fact that they havemultiple functional groups, is that they can be used as stable couplingagents, as described in patent application WO03/72546 filed by theApplicant. Another advantage also resides in the fact that they arestructures with multiple markers because their peripheral functionalgroups can be used to bind multiple markers such as biotin, fluorophoresor a combination thereof. Thus, patent application WO01/02861 describesdendrimers bound on the one hand to 1 to 1200 probes and on the otherhand to 1 to 1200 markers. These dendrimers are used for signalamplification.

Other polybiotinylated dendrimers have also been described in patentapplication WO00/72802.

However, conventional methods for preparing dendrimers have thedrawbacks that the dendrimers are synthesised in liquid phase, which ismore complex and more lengthy than in solid phase. Furthermore, somemethods produce non-neutral and non-polarised dendrimers.

The Applicant has now surprisingly developed a novel method forpreparing polybiotinylated dendrimer compounds which make it possible toovercome the drawbacks of the prior art methods in the sense that theyallow the reproducible and controlled preparation of the biotinylatedcompounds which are:

-   -   water-soluble although electrically neutral,    -   polarised thanks to a separate and precise spatial distribution        of their functions, including the biotin function, and    -   synthesised in solid phase.

Thus, a first object of the present invention is a method for preparinga compound having the formula (I):

where

X is biotin or

Y is biotin or

Z is biotin or

V is biotin or

B is NH₂ or H,

AA is a trifunctional molecule derivative when B is NH₂ or abifunctional molecule derivative when B is H,

AA₁ to AA₅ are each independently a trifunctional molecule derivative,

G₀ to G₅ are each independently an arm comprising at least one(—CH₂—CH₂—O) unit,

-   -   n₀ to n₅ are each independently a whole number between 1 and 8        and    -   T is an antiligand or a reactive group for fixation to an        antiligand, said antiligand being capable of reacting with a        ligand,        characterised in that it comprises the steps consisting in:        (i) grafting n₀ compounds having the formula W″₀-G₀-OH, where        W″₀ is an amine protecting group, n₀ and G₀ being such as        defined above, to a compound having the formula (II):

where R is a prefunctionalised resin, W and W₀ are different from oneanother and represent an amine protecting group, W being different fromW″₀, and AA is such as defined above,to obtain a compound having the formula (III):

(ii) coupling a compound having a formula (IV):

where W₁ and W′₁ are amine protecting groups that are identical to ordifferent from one another and from the W₀ and W″₀ groups, but differentfrom W, and AA₁ is such as defined above, with the compound having theformula (III) to obtain a compound having the formula (V):

(iii) grafting 2n₁ compounds having the formula W″₁-G₁-OH, where W″₁ isan amine protecting group, identical to or different from one anotherand from the other protecting groups used in this method, but differentfrom W, G₁ and n₁ being such as defined above, onto the compoundobtained in step (ii) to obtain a compound having the formula (VI):

(iv) when X in the formula (I) is biotin, passing directly to step (v);if not, when X in the formula (I) is not biotin, repeating the steps(ii) and (iii) with 2^(p-1) compounds having the formula (VII):

and 2^(p)n_(p) times the compound having the formula W″_(p)-G_(p)-OH,where p is a whole number between 2 and 5, and W_(p), W′p, W″_(p) areamine protecting groups that are identical to or different from oneanother and from the other protecting groups used in this method, butdifferent from W, and AA_(p) is a trifunctional molecule, according tothe following sequence:

-   -   1 time when Y is biotin, p then being equal to 2    -   2 times when Y is not biotin and Z is biotin, p then being equal        to 2 then to 3,    -   3 times when Y and Z are not biotin and V is biotin, p then        being equal to 2, 3 then 4, and    -   4 times when Y, Z and V are not biotin, p then being equal to 2,        3, 4 then 5,        (v) deprotecting the compound thus obtained at the W″₁ or W″_(p)        group, p being between 2 and 5, and coupling with biotin,        (vi) deprotecting, at the W group, the compound thus        polybiotinylated and coupling with an antiligand or a reactive        group for fixation to an antiligand (T) and        (vii) cutting the compound thus obtained from the resin (R) to        obtain a compound having the formula (I).

A further object of the invention relates to the compounds having theformula (I) in which:

X is biotin or

Y is biotin or

Z is biotin or

V is biotin or

B is NH₂ or H,

AA is a trifunctional molecule derivative when B is NH₂ or abifunctional molecule derivative when B is H,

AA₁ to AA₄ are each independently a trifunctional molecule derivative,

G₀ to G₅ are each independently an arm comprising at least one(—CH₂—CH₂—O—) unit,

n₀ to n₅ are each independently a whole number between 1 and 8 and

T is a maleimide group, a carboxylic acid group or an antiligand.

The invention finally relates to the use of the compounds that can beobtained by the method described above, for signal amplification in adiagnostic test.

The method of the invention therefore serves to prepare biotinylatedcompounds in a reproducible and controlled manner. Furthermore, saidcompounds obtained are highly soluble even without electrical charge dueto the presence of the substituents G₀ to G₅. Their particular dendrimerstructure allows polarisation of the molecule, of which the sterichindrance is controlled, thereby allowing a better presentation of thebiotins and hence the amplification of the signal, in particular when itis used in a diagnostic application, due to the presence of 2 to 32biotins. The compounds obtained are then particularly useful for thedetection of an analyte in a very small quantity.

The dendrimer structure of the compounds obtained by the method of theinvention is obtained thanks to the use of derivatives of trifunctionalor bifunctional molecules.

Bifunctional molecule is understood to mean any molecule comprising atleast one trivalent or tetravalent atom carrying two functions enablingthem to react with two molecules. These functions are selected from NH₂and COOH. These bifunctional molecules may then comprise two identicalor different functions, that is to say two NH₂ functions, two COOHfunctions or even one COOH function and one NH₂ function. As anon-limiting example of bifunctional molecules, mention can be made ofbifunctional diamines such as ethylenediamine and4,7,10-trioxamidecanediamine.

Derivative of bifunctional molecule is understood to mean thebifunctional molecule in which each of the two functions is in a bondingengagement with another chemical entity. Thus, the bifunctional moleculederivative consists of the skeleton of the bifunctional molecule inwhich each NH₂ function has lost a hydrogen atom and each COOH functionhas lost a hydroxyl (OH) radical. Thus, for example, when thebifunctional molecule derivative is ethylenediamine, having the formulaH₂N—CH₂—CH₂—NH₂, the ethylenediamine derivative is —HN—CH₂—CH₂—NH—.

When the substituent AA is a bifunctional molecule derivative, B is H inthe formula (I).

Trifunctional molecule is understood to mean any molecule comprising atleast one trivalent or tetravalent atom carrying three functionsenabling them to react with three molecules. These functions areselected from NH₂ and COOH. These trifunctional molecules may thencomprise three identical or different functions, that is to say, threeNH₂ functions, two NH₂ functions and one COOH function, two COOHfunctions and one NH₂ function or even three COOH functions. As anon-limiting example of trifunctional molecules, mention can be made oflysine, diaminobutanoic acid, diaminopropanoic acid, L-ornithine andderivatives thereof.

Trifunctional molecule derivative is understood to mean thetrifunctional molecule in which each of the three functions is in abonding engagement with another chemical entity. Thus, the trifunctionalmolecule derivative consists of the skeleton of the trifunctionalmolecule in which each NH₂ function has lost a hydrogen atom and eachCOOH function has lost a hydroxyl (OH) radical. Thus, for example, whenthe trifunctional molecule derivative is lysine, having the formulaH₂N—(CH₂)₄—CH(NH₂)—COOH, the lysine derivative is—HN—(CH₂)₄—CH(NH—)—CO—.

When the substituent AA is a trifunctional molecule derivative, B is NH₂in the formula (I).

According to a preferred embodiment, the compounds in the method of theinvention have one of the following features:

the substituents AA₁ and AA₂, if applicable AA₃, AA₄ and AA₅, areidentical and are preferably a lysine derivative,

the substituent AA is a lysine derivative and B is NH₂.

The solubility of the compounds obtained with the method of theinvention derives from the nature of the spacer arms G₀ to G₅ usedbecause they comprise at least one (—CH₂—CH₂—O—) unit. Preferably, thecompounds of the invention comprise one to six (—CH₂—CH₂—O—) units, evenmore preferably one to four (—CH₂—CH₂—O—) units. As non-limitingexamples of such spacer arms, mention can be made ofpentaoxaoctadecanoyl, tetraoxapentadecanoyl, trioxadodecanoyl,trioxamidecanoyl, dioxaoctanoyl, oxapentoyl and hexaoxaheneicosanoyl andderivatives thereof.

According to an embodiment, the compounds in the method of the inventionhave one of the following features:

the substituents G₀, G₁, if applicable G₂, G₃, G₄ and G₅ are identicaland preferably have the formula (—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—).

the whole numbers n₀, n₁, if applicable n₂, n₃, n₄ and n₅, are identicaland are preferably equal to 2 or 3.

In the compounds prepared according to the method of the invention, T isan antiligand or a reactive group for fixation to an antiligand.

Antiligand is understood to mean any molecule capable of bonding with aligand. Examples of ligand/anti-ligand pairs are well known to theperson skilled in the art, which is the case for example with thefollowing pairs: haptene/antibody, antigen/antibody, peptide/antibody,sugar/lectin, polynucleotide/polynucleotide complementary.

Reactive groups for fixation to an antiligand are widely known to theperson skilled in the art. Non-limiting examples of such groups are themaleimide, carboxylic acid, thiol, amine, alkoxyamine, hydrazine, azido,alkyne, aldehyde groups.

The compounds obtained by the method according to the invention allowthe amplification of the detection signal in a diagnostic test due tothe presence of several biotins, according to Table 1 below:

TABLE 1 Number of biotins X Y Z V  2 Biotin NA NA NA  4

biotin NA NA  8

biotin NA 16

biotin NA: not applicable

According to a particular embodiment of the invention, X is

and Y is biotin.

The grafting and coupling steps of the method according to the inventionare steps conventionally used by the person skilled in the art in thefield of solid-phase synthesis of the peptide synthesis type. Thesesteps can be implemented manually or even in an automated manner oncommercial synthesisers such as the ABI 431 A and ABI433A synthesiser.

The reagents used during these steps are known to the person skilled inthe art and are described for example in Chemical Approaches to theSynthesis of Peptides & Proteins, Paul Lloyd Williams, FernandoAlbericio, Ernest Giralt, CRC Press.

Thus, the compounds having the formula (II) in which R is aprefunctionalised resin and AA is a bifunctional molecule derivative areavailable from Novabiochem. By way of example, mention can be made of:

-   -   the compound Universal NovaTag resin, reference 04-12-3910        having the formula:

-   -   the compound Universal PEG NovaTag resin, reference 04-12-3911        having the formula:

When AA is a trifunctional molecule in the compounds having formula(II), the latter can be obtained by coupling a compound having theformula (II′)

in which W_(o) and W are such as defined above, onto a solid amine phasehaving the formula (II″) W_(r)—R in which R is

and W_(r) is an amine protecting group identical to or different toW_(o) and W.

An example of a solid amine phase comprises the resin RINK-amide-MHBAfrom Novabiochem (Reference 01-64-0037).

The steps of deprotection and separation of the polybiotinylatedcompound of the resin R are widely known to the person skilled in theart and are described for example in Chemical Approaches to theSynthesis of Peptides & Proteins (supra).

Amine protecting groups are also widely known to the person skilled inthe art and, as non-limiting examples, mention can be made oft-butyloxycarbonyl (Boc), benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl(Fmoc) or methoxytrityl (Mmt). Any protecting group can be employed inthe method according to the invention insofar as W is different from theother protecting groups used.

According to an embodiment, the compounds in the method according to theinvention have one of the following features:

the W group is methoxytrityl,

the W₁ and W′₁ groups are identical and, if applicable, W₂ and W′₂ areidentical, W₃ and W′₃ are identical, W₄ and W′₄ are identical and W₅ andW′₅ are identical,

the W₁, W′₁ groups and, if applicable, W₂, W′₂, W₃, W′₃, W₄ and W′₄, W₅and W′₅ are fluorenylmethoxycarbonyl groups.

The method according to the invention serves to prepare novelpolybiotinylated dendrimer compounds having the formula (I)

where

X is biotin or

Y is biotin or

Z is biotin or

V is biotin or

B is NH₂ or H,

AA is a trifunctional molecule derivative when B is NH₂ or abifunctional molecule derivative when B is H,

AA₁ to AA₄ are each independently a trifunctional molecule derivative,

G₀ to G₅ are each independently an arm comprising a (—CH₂—CH₂—O—) unit,

n₀ to n₅ are each independently a whole number between 1 and 8 and

T is a maleimide group, a carboxylic acid group or an antiligand.

According to a particular embodiment, the compounds of the inventioncomprise one of the following features:

the substituents AA₁ and AA₂, if applicable AA₃, AA₄ and AA₅, areidentical and are preferably a lysine derivative,

the substituents G₀, G₁, if applicable G₂, G₃, G₄ and Gs, are identicaland preferably have the formula (—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—),

the whole numbers n₀, n₁, if applicable n₂, n₃, n₄ and n₅, are identicaland are preferably equal to 2 or 3,

X is

and Y is biotin,

AA is a lysine derivative and B is NH₂,

the antiligand is a Fab′ fragment,

X is

Y is biotin, AA, AA₁ and AA₂ are a lysine derivative, G₀, G₁ and G₂ havethe formula (—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—), n₀, n₁ and n₂ are equalto 2, B is NH₂ and T is an antiligand, preferably a Fab′ fragment.

The compounds prepared by the method of the invention are particularlyuseful for signal amplification in in vitro diagnostic methods implyinga recognition of a ligand/anti-ligand pair, the molecule to be diagnosedconstituting the ligand, which thus constitutes another object of theinvention.

In fact, the compounds of the invention directly possess an antiligand(substituent T) or are reacted with an antiligand which shall be fixedon said substituent T by methods well known to the person skilled in theart.

Examples of ligand/anti-ligand pairs are well known to the personskilled in the art, which is the case for example of the followingpairs: haptene/antibody, antigen/antibody, peptide/antibody,sugar/lectin, polynucleotide/polynucleotide complementary.

In vitro diagnostic methods which can employ the conjugates of theinvention are in particular immunological tests such as the sandwichmethods like ELISA, IRMA and RIA, methods called competition methods anddirect immune detection methods such as immunohistochemistry,immunocytochemistry, Western-blot and Dot-blot. All these methods arewell known to the person skilled in the art.

Diagnostic methods employing compounds prepared according to the methodof the invention are useful both in clinical diagnosis of diseases andin the diagnosis of products prepared in industry (industrial diagnosis)such as products intended for the agroprocessing industry and productsintended for health care.

Diseases which can be diagnosed with the compounds of the invention arenot limited and comprise all diseases revealed by the presence of aspecific marker of the disease, such as the molecule of biologicalinterest or analyte or ligand, for which a bonding partner exists. Asnon-limiting examples, mention can be made of viral diseases such ashepatitis and AIDS, and solid cancers such as breast, colon or prostatecancer.

The signal amplification is carried out by the use of a marker moleculecapable of directly or indirectly generating a detectable signal. Anon-limiting list of these markers consists of:

-   -   enzymes which produce a signal that is detectable for example by        colourimetry, fluorescence, luminescence, such as horseradish        peroxidase, alkaline phosphatase, β-galactosidase,        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent, luminescent, dye compounds,    -   radioactive molecules such as ³²P, ³⁵S or ¹²⁵I, and    -   fluorescent molecules such as Alexa or phycocyanines,

with the understanding that these markers will be modified to be boundto a bonding partner with biotin such as streptavidin. This type ofmodification is well known to the person skilled in the art.

According to the type of marking used, the person skilled in the artwill add reagents allowing visualisation of the marking.

The invention will be better understood with the help of the followingexamples which are provided for illustration and are non-limiting, andwith reference to FIGS. 1 to 8, in which:

FIG. 1 corresponds to the developed formula of a compound having 4biotins according to an embodiment,

FIG. 2 is a graphic representation giving the OD results of a directELISA test of an anti-Listeria monocytogenes Fab′ conjugate that ismonobiotinylated (by biotin BMCC) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of amicroplate, according to its concentration,

FIG. 3 is a graphic representation giving the OD results of a directELISA test of an anti-Salmonella Fab′ conjugate that is monobiotinylated(by biotin BMCC) or tetrabiotinylated (by a compound according to anembodiment of the invention), fixed to the bottom of a microplate,according to their concentration, and also an indirect ELISA test withthese same conjugates, but detected by means of Salmonella antigens,

FIG. 4 is a graphic representation giving the OD results of a directELISA test of anti-HIV Fab′ conjugate that is monobiotinylated (bybiotin BMCC) or tetrabiotinylated (by a compound according to anembodiment of the invention), fixed to the bottom of a microplate,according to its concentration,

FIG. 5 is a graphic representation giving the OD results of a directELISA test of an anti-Kallikrein Fab′ conjugate that is monobiotinylated(by biotin BMCC) or tetrabiotinylated (by a compound according to anembodiment of the invention), fixed to the bottom of a microplate,according to its concentration,

FIG. 6 is a graphic representation giving the OD results of a directELISA test of an antigen gp160 conjugate reduced to DTT that ismonobiotinylated (by biotin BMCC) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of amicroplate, according to its concentration,

FIG. 7 is a graphic representation giving the OD results of a directELISA test of surface antigen conjugates of hepatitis B that aremonobiotinylated (by biotin NHs) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of amicroplate, according to their concentration, and

FIG. 8 is a graphic representation giving the OD results of a directELISA test of an anti-Salmonella antibody conjugate that ismonobiotinylated (by biotin NHs) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of amicroplate, according to its concentration.

EXAMPLE 1 Preparation of a Compound Having the Formula (I) with 4Biotins

(AA=AA₁=AA₂=lysine derivative;

B is NH₂; G₀=G₁=G₂=(—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—)=Ado;

n₀=n₁=n₂=2T=maleimide

X=

Y=biotin)

About 132 μmoles of Fmoc-RINK-MBHA (Novabiochem) resin were placed in areactor of a 433A automatic synthesiser (Applied Biosystems) equippedwith a UV detector. Cartridges were also prepared each containingsynthons at the rate of 1 mmole per cartridge:

-   -   Fmoc-Lys(Mmt)-OH, 640.8 mg (Novabiochem)    -   Fmoc-Lys(Fmoc)-OH, 590.7 mg (Novabiochem)    -   Fmoc-Ado-OH, 385.4 mg (or Fmoc-8-amino-3,6-dioxaoctanoic acid,        Polypeptide)    -   N-maleoyl-βalanine, 169.1 mg (FLUKA)    -   Biotin, MW 244.3 (SIGMA) previously taken up in 0.5 M solution        in DMSO (ALDRICH), or 2 ml of solution per cartridge        The following raw materials were used:    -   N-methyl-pyrrolidone or NMP (Applied Biosystems)    -   Dichloromethane or DCM (Applied biosystems)    -   Piperidine (ALDRICH)    -   Acetic anhydride (FLUKA)    -   Methanol (MERCK)    -   Solution of di-isopropylethylamine or DIEA (ALDRICH), 2 M in NMP        above    -   Solution of hexafluorophosphate benzotriazolyl        tetramethyluronium or HBTU (Novabiochem) in equivalence with        N-hydroxybenzotriazole (SENN) the whole in solution at about        0.45 M in N,N-dimethylformamide or DMF (ALDRICH)    -   Solution containing 1% trifluoroacetic acid or TFA (ACROS) and        2.5% tri-isopropylsilane or TIS (FLUKA) in the same DCM as        above.

By default, 2 cartridges of each synthon were provided to beincorporated in order to work in Conditional Double Coupling mode.

The programming of the synthesiser is based on a succession of cycles,themselves divided into modules, the modules being a series of primaryfunctions on the programmable logic controller. The programmes of thesupplier were used as the basis, and were adapted for the preparation ofthe following example:

The following molecules were used:

-   -   A: recovery of the amino acid powder cartridge    -   B: deprotection of the Fmoc N-terminal group with piperidine    -   C: capping or masking of unreacted amines with acetic anhydride    -   D: rinsings of the resin with NMP    -   E: activation and transfer of the amino acid solution to the        resin    -   F: coupling with stirring    -   G: deprotection of the monomethoxytrityl of the side chain of        the 1^(st) lysine    -   I: intermittent stirring of the reactor for 30 minutes    -   a: conditional module for recovery of the cartridge, rinsing of        the resin and activation/transfer to the reactor    -   b: conditional module of additional deprotections with        piperidine    -   c: rinsing of the resin with DCM    -   d: other module of rinsing of the resin with NMP    -   f: conditional module of long coupling with stirring    -   i: conditional module of ejection of unused cartridge        The synthesiser was programmed according to the cycles as        indicated in Table 2 below:

TABLE 2 Cycle AA Name Modules 1 Fmoc-Lys(Mmt)-OH Conditional doublecoupling and BbADEFfiafCd capping 2 Fmoc-Ado-OH Conditional doublecoupling and BbADEFfiafCd capping 3 Fmoc-Ado-OH Conditional doublecoupling and BbADEFfiafCd capping 4 Fmoc-Lys(Fmoc)- Conditional doublecoupling and BbADEFfiafCd OH capping 5 Fmoc-Ado-OH Long condition doublecoupling and BbADEFfIiafICd capping 6 Fmoc-Ado-OH Long conditionaldouble coupling and BbADEFfIiafICd capping 7 Fmoc-Lys(Fmoc)- Longconditional double coupling and BbADEFfIiafICd OH capping 8 Fmoc-Ado-OHVery long conditional double coupling BbADEFfIIiafIICd and capping 9Fmoc-Ado-OH Very long conditional double coupling BbADEFfIIiafIICd andcapping 10 Biotin Very long conditional double coupling BbADEFfIIiafIICdand capping 11 N-maleoyl-βalanine Long double coupling Lys(Mmt)GcADEFIAdEFfI deprotection and capping Cd 12 None Resin washings withNMP and DCM Dcc

During the deprotections of the Fmoc group, the apparatus performs atest. According to the result obtained, the programme makes a single ordouble coupling (use respectively only 1 or the 2 cartridges provided).

1.1. Coupling 1^(st) lysine (AA)

The resin is deprotected and Fmoc-Lys(Mmt)-OH (II′) is coupled asfollows (cycle 1: the apparatus uses only 1 or the 2 cartridges ofFmoc-Lys(Mmt)-OH amino acid according to the result of the conditionaltest):

(1) or (II″): also represented by

1.2. Construction of the Skeleton:

This consists in the alternation of:

-   -   a) grafting the hydrophilic spacer arm Ado₂    -   b) coupling the branching synthon=Fmoc-Lys(Fmoc)-OH        Based on (1) above        i) grafting of Hydrophilic Spacer Arm Ado₂ (Step a)        (cycles 2 and 3: the apparatus uses 1 or the 2 cartridges of        Fmoc-Ado-OH amino acid in each cycle according to the result of        the conditional tests)

ii) Branching by Fmoc-Lys(Fmoc)-OH (Step b)

(cycle 4: the apparatus uses only 1 or the 2 cartridges ofFmoc-Lys(Fmoc)-OH amino acid according to the result of the conditionaltest)

Based on (3)

iii) Repetition of Steps a), b) then a to Terminate the Skeleton

cycles 5 and 6: the apparatus uses 1 or the 2 cartridges of eachFmoc-Ado-OH amino acid according to the result of the conditional tests:

cycle 7: the apparatus uses only 1 or the 2 cartridges ofFmoc-Lys(Fmoc)-OH amino acid according to the result of the conditionaltest:

cycles 8 and 9: the apparatus uses only 1 or the 2 cartridges ofFmoc-Ado-OH amino acid according to the result of the conditional tests:

1.3. Coupling of a Biotin to Each End of the 4 Branches:

(cycle 10: the apparatus uses only 1 or the 2 cartridges of biotinaccording to the result of the conditional test)

1.4. Deprotection of Mmt and Grafting of Maleimide:

(cycle 11: this time, the programme uses the 2 cartridges ofN-maleoyl-βalanine—no conditional test).

The protecting group Mmt is cut selectively by repeated action of asolution of DCM (dichloromethane) containing 1.5% TFA (trifluoroaceticacid) and 1.5% TIS (tri-isopropylsilane). The amine of the side chain ofthe lysine inserted in step 1) is thus liberated for its coupling withan acidic form of maleimide, N-maleoyl-β-alanine.

1.5. Cleavage

After synthesis, the resin is dried by nitrogen blanketing. It istransferred to a 20 ml plastic syringe comprising a sinter and contactedwith 10 ml of a TFA/water (95/5) solution with stirring for 1 hour 30minutes at ambient temperature. The cleavage solution is then collectedby filtration (via the sinter of the syringe) in a glass flask. Theresin is rinsed with about 5 ml of TFA, and then 3 times with about 5 mlof DCM. All these volumes are added to the flask. The raw cleavagemixture is evaporated under reduced pressure with a rotary evaporator ofwhich the bath is at ambient temperature for a few tens of minutes. Anoily orange-coloured residue is finally obtained.

This product is taken up in a few millilitres of deionised water foranalyses and purification.

The aliquot, rediluted in deionised water, is analysed by HPLC and massspectrometry coupled to HPLC.

BECKMAN analytical HPLC with VYDAC C₁₈ reverse-phase chromatographycolumn 5.4 mm in diameter by 250 mm in length, with the eluants A=watercontaining 0.1% TFA and B=mixture of acetonitrile or ACN with water(95/5 respectively) containing 0.1% TFA. The flow rate is 1 ml/min.Sample injection is followed by a gradient from 0 to 80% acetonitrile in30 minutes. Under these conditions, after the dead volume peak at about3 minutes, the tetrabiotinylated molecule/maleimide exits at around 15minutes. LC/MS analysis (HPLC and THERMO ELECTRON mass detector) is usedto identify the compound anticipated with a recalculated molecularweight of 3617.7 g/mole (theoretical 3618.26)

The remainder of the raw mixture is purified in at least threeequivalent volumes by injection in BECKMAN semi-preparative HPLC on aVYDAC C₁₈ reverse-phase column 20 mm in diameter by 250 mm in length, atthe rate of 22 ml/min. The eluants

A and B are identical to those used in analytical HPLC. Typicalpurification programme: after injection, rest for 10 minutes at 5%eluant B; then passage from 5 to 23% eluant B in 1 minute; thenpurification gradient from 23 to 31% B in 30 minutes. Under theseconditions, the purification fractions are taken on arrival of the bulkof the desired product, from about 21 minutes, at the rate of 0.33minute per tube, until about minutes (about 25 tubes). An aliquot ofeach tube is analysed in isocratic mode by analytical HPLC (sameconditions as above except isocrate of 26% eluant B for 10 minutes).

The purest fractions are collected in a glass flask and the solution isthen freeze-dried. After freeze-drying and weighing, the lyophilisate istaken up in deionised water at 1 mg/ml according to the weighing. Thisstock solution is distributed in brown glass bottles at the level of 1to a few millilitres per bottle. The bottles are then again freeze-driedand plugged under inert atmosphere of 700 millibar argon.

An aliquot is used to perform the final analysis by HPLC (BECKMAN,identical conditions to the 1^(st) analysis with gradient above), LC/MS(conditions identical to above) and analysis of amino acids according tothe supplier's procedure on the Agilent 1100 system series withdetection by fluorescence.

The tetrabiotinylated compound (13) thus obtained has the developedformula as described in FIG. 1.

EXAMPLE 2 Preparation of a Compound Having the Formula (I) with 4Biotins

(AA=AA₁=AA₂=lysine derivative;

B is NH₂; G₀=G₁=G₂=(—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—)=Ado;

n₀=n₁=n₂=3T=maleimide

X=

Y=biotin)

In this example, the same reagents were used as those described inexample 1, with the exception of the resin: about 148 micromolecules ofH-RINK-ChemMatrix resin (MATRIX Innovation) in free amine form withoutFmoc group were placed in the reactor of the ABI433A synthesiser.

2.1 Coupling 1^(st) lysine (AA):

Unlike in example 1, Fmoc-Lys(Mmt)-OH was coupled manually. The resin isalready in amine form and does not require deprotection. 137 micromolesof Fmoc-Lys(Mmt)-OH taken up in solution in NMP were then sent manuallyto the reactor with 1 equivalent of HBTU/HOBt coupling agent. Thereagents finally have a volume of about 6 ml. The coupling reaction isthen started up by sending 0.5 ml of the same solution of DIEA 2 M/NMPinto the reactor. The resin is in slight excess. The reaction is carriedout for 2 hours at laboratory temperature with intermittent vortex. Thesynthesis is then resumed in fully automated mode following theprogramme indicated in Table 3 below:

TABLE 3 Cycle AA Name Modules 1 Fmoc-Lys(Mmt)-OH End of coupling andmasking of residual IIDCCDD amines of the resin 2 Fmoc-Ado-OHConditional double coupling and BbADEFfiafCd capping 3 Fmoc-Ado-OHConditional double coupling and BbADEFfiafCd capping 4 Fmoc-Ado-OHConditional double coupling and BbADEFfiafCd capping 5 Fmoc-Lys(Fmoc)-Conditional double coupling and BbADEFfiafCd OH capping 6 Fmoc-Ado-OHLong conditional double coupling and BbADEFfIIiafIICd capping 7Fmoc-Ado-OH Long conditional double coupling and BbADEFfIIiafIICdcapping 8 Fmoc-Ado-OH Long conditional double coupling andBbADEFfIIiafIICd capping 9 Fmoc-Lys(Fmoc)- Long conditional doublecoupling and BbADEFfIIiafIICd OH capping 9 Fmoc-Ado-OH Very longconditional double coupling BbADEFfIIIIiafII and capping IICd 10Fmoc-Ado-OH Very long conditional double coupling BbADEFfIIIIiafII andcapping IICd 11 Fmoc-Ado-OH Very long conditional double couplingBbADEFfIIIIiafII and capping IICd 12 Biotin Very long conditional doublecoupling BbADEFfIIIIiafII and capping IICd 13 N-maleoyl-βalanineDeprotection Lys(Mmt) long double GcADEFIAdEFfI coupling and capping Cd14 None Resin washing with NMP and DCM Dcc

After the first cycle of this programme, the same type of structure isobtained as compound (I) of example 1.

2.2 Construction of the Skeleton

The procedure described in section 1.2 step i) was repeated, except thatthis step i) is repeated three times and in consequence, 3 Fmoc-Ado-OH(and no longer 2) are coupled in succession to achieve the followingstructure (step 2.2.i)):

The branching was then carried out identically to step 1.2 ii) bycoupling Fmoc-Lys(Fmoc)-OH to obtain (step 2.2.ii)):

Steps 2.2 i) and 2.2 ii) were then repeated identically to achieve thefollowing molecule with two branches:

Steps 2.2 i) and 2.2 ii) above were then repeated identically again toachieve the intermediate molecule with four branches protected by“Fmoc”:

The procedure described in sections 1.3, 1.4 was then followed toachieve the tetrabiotinylated molecule with maleimide:

The cleavage of the molecule from its support, the analysis andpurification, were then carried out under conditions similar to those ofexample 1. With analytical HPLC (identical experimental conditions) theexit of the tetrabiotinylated molecules/maleimide was observed with armsof “3 Ado” at around 15 minutes. The LC/MS analysis (identicalconditions) confirms the presence of the anticipated compound with arecalculated molecular weight of 4634 g/mole (theoretical 4634.37). Thismolecule was purified under identical conditions to example 1, exceptthat the purification gradient consists this time of a passage from 22to 32% of eluant B, also in 30 minutes.

As in example 1, the purest fractions are collected and the mixture isfreeze-dried. After this, the lyophilisate is weighed and taken up inwater (soluble) and distributed in fractions of 1 to a few millilitres,which are freeze-dried in turn and then plugged under inert atmosphereof 700 millibar argon.

As in example 1, an aliquot is used to perform the identical finalanalyses.

EXAMPLE 3 Preparation of a Compound Having the Formula (I) with 8Biotins

(AA=AA₁=AA₂=AA₃=lysine derivative;

B is NH₂;

G₀=G₁=G₂=G₃=(—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—)=Ado;n₀=n₁=n₂=3T=maleimide

Y=

Z=biotin

To obtain this structure with eight branches, the method use in example1 was repeated, with a few differences. The programme used on theABI433A synthesiser is the one shown in Table 4 below:

TABLE 4 Cycle AA Name Modules 1 Fmoc-Lys(Mmt)-OH Single coupling in lowstoechiometry BbDgFfIIIIIICCd and masking of residual amines of theresin 2 Fmoc-Ado-OH Conditional double coupling and BbADEFfiafCd capping3 Fmoc-Ado-OH Conditional double coupling and BbADEFfiafCd capping 4Fmoc-Ado-OH Conditional double coupling and BbADEFfiafCd capping 5Fmoc-Lys(Fmoc)- Conditional double coupling and BbADEFfiafCd OH capping6 Fmoc-Ado-OH Conditional double coupling and BbADEFfiafCd capping 7Fmoc-Ado-OH Conditional double coupling and BbADEFfiafCd capping 8Fmoc-Ado-OH Conditional double coupling and BbADEFfiafCd capping 9Fmoc-Lys(Fmoc)- Conditional double coupling and BbADEFfiafCd OH capping9 Fmoc-Ado-OH Long conditional double coupling and BbADEFfIIiafIICdcapping 10 Fmoc-Ado-OH Long conditional double coupling andBbADEFfIIiafIICd capping 11 Fmoc-Ado-OH Long conditional double couplingand BbADEFfIIiafIICd capping 12 Fmoc-Lys(Fmoc)- Long conditional doublecoupling and BbADEFfIIiafIICd OH capping 13 Fmoc-Ado-OH Long conditionaldouble coupling and BbADEFfIIIAdE capping FfIIICd 14 Fmoc-Ado-OH Longconditional double coupling and BbADEFfIIIAdE capping FfIIICd 15Fmoc-Ado-OH Long conditional double coupling and BbADEFfIIIAdE cappingFfIIICd 16 Biotin Long conditional double coupling and BbADEFfIIIAdEcapping FfIIICd 17 N-maleoyl-βalanine Deprotection Lys(Mmt) long doubleGcADEFIAdEFfI coupling and capping Cd 18 None Resin washings with NMPand DCM Dcc

The loading of 448 micromoles of resin (Fmoc-RINK-MBHA) is carried outautomatically but with only 80 micromoles of Fmoc-Lys(Mmt)-OH to achievethe compound on resin (1) of example 1. The excess amine on the resin ismasked by acetylation.

The synthesis then continues as an example 2, until the step forobtaining the structure (17).

From then on, steps 2.2 i) and 2.2 ii) of example 2 are repeatedidentically, to achieve the intermediate molecule with eight branchesprotected by “Fmoc” groups:

Then, as for example 1, identical steps to sections 1.3, 1.4 of example1 were carried out, to achieve the octobiotinylated molecule withmaleimide:

The cleavage of the molecule from its support is carried out undersimilar conditions to example 1.5, as well as the analysis andpurification. In analytical HPLC (identical experimental conditions),the octobiotinylated molecule/maleimide with arms of 3 Ado exits in theform of a broad peak at around 16 minutes. The LC/MS analysis (identicalconditions) confirms the presence of the anticipated compound with arecalculated molecular weight of 9535 g/mole (theoretical 9536.06). Thismolecule is purified under identical conditions to example 1, exceptthat the purification gradient consists of a passage from 25 to 35% ofeluant B in 30 minutes.

As in example 1, the purest fractions are collected and the mixture isfreeze-dried. After this, the lyophilisate is weighed and taken up inwater (soluble) and distributed in fractions of 1 to a few millilitres,which are freeze-dried in turn and then plugged under inert atmosphereof 700 millibar argon.

As in example 1, an aliquot is used to perform the identical finalanalyses.

The octobiotinylated molecule/maleimide with arms of “3 Ado” obtained isthe following molecule (22):

EXAMPLE 4 Coupling of SH Proteins with a Compound Having the Formula (I)

4.1. Context

The coupling of biotin on SH proteins (for example Fab′ fragments,antigens reduced with DTT and proteins modified by Traut's reagent) iscarried out conventionally by using biotin BMCC (Pierce ref21900:1-Biotinamido-4{4′-(maleimidomethyl)cyclohexanecarboxamido]butane)with fixes to the free sulphydryl functions of the SH proteins. However,the small quantity of free sulphydryl functions does not allow fixationof a large number of biotins, so that when the biotin/SH proteinconjugate is used for diagnosis, the detection signal is weak, which isrepresentative of the presence of one biotin per conjugate. It istherefore important to enhance the signal.

The compounds of the invention serve to bond from 2 to 32 biotins andthe Applicant has demonstrated that, against all expectations, theypreserved a signal proportional to the number of biotins present, aftercoupling the SH proteins to the free sulphydryl radicals, despite theirsteric hindrance due to the large number of bonded biotins.

The coupling protocol is described in the following sections.

4.2. Reagents

The commercial reagents used are listed in Table 5.

TABLE 5 Name Supplier References Pepsin agarose Sigma P0609-50KU 2 βmercapto ethanol Thermo scientific 35602 Biotin BMCC Thermo scientific21900 DMSO Merck 1.02950.0500 Iodoacetamide Sigma I6125 Traut's reagentThermo scientific 26101 N-ethylmaleimide Sigma E-3876 DTT Sigma D0632Biotin NHs Roche 11003933

Furthermore, the SH proteins used, obtained from bioMerieux, France, arethe following:

-   -   Fab′ fragments: anti-Listeria monocytogenes Fab′ fragment,        anti-antigen of the Salmonella wall, anti-p24 of HIV clone A and        clone B and anti-human kallikrein 2;    -   Antigens used: gp160 of HIV, HBs Ad of HBV, HBs Ay of HBV    -   Antibodies: Salmonella wall anti-antigen antibody.

The antigens used for the indirect ELISA assay of the Fab′ fragments ofSalmonella were obtained after culture of Salmonella bergedorf,typhimurium and enteritidis strains in peptone medium for 24 h at 37°C., and then heating for 15 min at 100° C.

Finally, the compound having formula (I) prepared according to example 1was used.

4.3. Coupling to Biotin BMCC and to the Compound of Example 1 on Fab′

The conjugates were obtained by coupling as follows:

Fab′ fragment obtained by pepsic digestion, then reduction with 2βME,adjusted to 2 mg/ml after purification on Superdex 200 prep grade, inPO₄50 mM+NaCl 150 mM+EDTA(4Na) buffer 5 mM pH=6.8,

preparation of a freshly prepared solution of biotin BMCC (533.69g/mole) at 8.5 mM (4.54 g/l) in DMSO,

preparation of a freshly prepared mixture of compound of example 1(3618.26 g/mole) containing 2.5 mg/ml of deionised water,

coupling to Fab′ at the rate of 5 moles biotin BMCC or compound ofexample 1 per mole of Fab′ (46,000 Daltons),

incubation for 2 hours at laboratory temperature with rotary stirring ina brown glass bottle,

blockage by addition of equimolar iodoacetamide to the biotin BMCC or tothe compound of example 1,

use of a freshly prepared solution of 10 mM iodoacetamide in PBS buffer,

incubation for 1 h at laboratory temperature with rotary stirring,

dialysis against PBS buffer+azide,

exit from dialysis and determination of the concentration of theconjugates by measurement of OD at 280 nm (E=1.48)

4.4. Coupling of the Compound of Example 1 onto Antibodies Modified byTraut's Reagent

The conjugates were obtained by coupling as follows:

modification of the antibody by addition of 20 moles of Traut's reagentfor one mole of antibody, then dialysis in PO₄ 50 mM+NaCl 150mM+EDTA(4Na) 5 mM buffer, pH=6.8,

preparation of a freshly prepared solution of compound of example 1(3618.26 g/mole) at 2.5 mg/ml in deionised water,

coupling to the modified antibody at the rate of 20 moles of compound ofexample 1 per mole of antibody (160,000 Daltons),

incubation for 2 hours at laboratory temperature with rotary stirring ina brown glass bottle,

blockage by addition of equimolar iodoacetamide to the compound ofexample 1,

use of a freshly prepared solution of iodoacetamide in PBS,

incubation for 1 h at laboratory temperature with rotary stirring,

dialysis against PBS buffer+azide,

exit from dialysis and determination of the conjugate concentration bymeasurement of the OD at 280 nm (E=1.4)

4.5. Coupling of the Compound of Example 1 on Antigens Modified byTraut's Reagent

The conjugates were obtained by coupling as follows:

modification of the antigen by addition of 4 moles of Traut's reagentfor 1 mole of antigen, then dialysis in PO₄ 50 mM+NaCl 150 mM+EDTA(4Na)5 mM buffer, pH=6.8,

preparation of a freshly prepared solution of compound of example 1(3618.26 g/mole) at 1 mg/ml in deionised water,

coupling to the modified antigen at the rate of 1 moles of compound ofexample 1 per mole of antigen (MW=25,000 Daltons),

incubation for 2 hours at laboratory temperature with rotary stirring ina brown glass bottle,

blockage by addition of equimolar iodoacetamide to the compound ofexample 1,

use of a freshly prepared solution of iodoacetamide in PBS,

incubation for 1 h at laboratory temperature with rotary stirring,

dialysis against PBS buffer+azide+SDS (sodium dodecylsulphate),

exit from dialysis and determination of the concentration of theconjugates by measurement of the OD at 280 nm.

4.6. Coupling of Biotin NHs onto Antigens or Antibodies

The conjugates were obtained by coupling as follows:

dialysis of the antibodies or antigens in 0.1M NaHCO₃ buffer pH=8.3

preparation of a freshly prepared solution of biotin NHs at 11.36 g/l inDMSO for coupling the antibodies and a solution of 2.0 g/l in DMSO forcoupling the antigens

coupling to the antibody at the rate of 20 moles of biotin NHs per moleof antibody (160,000 Daltons), and coupling to the antigen at the rateof 1 mole biotin NHs per mole of antigen (25,000 Daltons)

incubation for 1 hour at ambient temperature with rotary stirring in abrown glass bottle,

blockage by addition of equimolar 1M lysine pH=8.0 to the biotin NHs,

incubation for 1 h at ambient temperature with rotary stirring,

dialysis against PBS buffer+azide for the antibodies and inPBS+azide+SDS for the antigens,

exit from dialysis and determination of the conjugate concentration bymeasurement of OD at 280 nm.

4.7. Coupling of the Compound of Example 1 and Biotin BMCC onto AntigensReduced with DTT

The conjugates were obtained by coupling as follows:

reduction of the antigen by addition of 800 moles of DTT per mole ofantigen and stirring for 30 min at laboratory temperature,

desalting of the reduced antigen on Sephadex G25 gel in PBS buffer+EDTApH=7.5,

preparation of a freshly prepared solution of biotin BMCC (533.69g/mole) at 4.35 mM (2.32 g/l) in DMSO,

preparation of a freshly prepared solution of the compound of example 1(3618.26 g/mole) at 2.5 mg/ml in deionised water,

coupling to the reduced antigen at the rate of 20 moles of biotin BMCCor of the compound of example 1 per mole of antigen (160,000 Daltons),

incubation for 2 hours at laboratory temperature with stirring on vortexin a brown glass bottle,

blockage by addition of 20 moles of NEM per mole of antigen,

use of a freshly prepared solution of 10 mM of NEM in PBS buffer,

dialysis of the conjugates in PBS+MIT,

exit from dialysis.

EXAMPLE 5 Application to ELISA Direct and Indirect Detection Tests

5.1. Direct ELISA Test

The activity of the conjugates comprising a tetrabiotinylated compoundof the invention or a single biotin is compared with a direct ELISAtest, by fixing the conjugates onto microplate and by developing thesignal by means of streptavidin coupled to peroxidase. This test servesto compare the “coupling level” of the polybiotinylated compounds,according to an embodiment of the invention, with monobiotinylatedcompounds.

Protocol:

Use of Maxisorp well strips (for deposition of antibody fragments andwhole antibodies) or Polysorp well strips (for deposition of antigens).Dilution of the monobiotinylated or tetrabiotinylated conjugates between0.05 μg/ml and 0.5 μg/ml in 50 mM CO₃ buffer pH=9.6, followed by twofolddilutions.Deposition of 100 μl/well of each dilutionIncubation overnight at laboratory temperature3 washings in PBS tweenDeposition of 100 μl/well of streptavidin-peroxidase diluted to 1/60000in PBSIncubation for 15 min in the oven at 37° C.3 washings in PBS tweenDeposition of 100 μl/well OPDIncubation for 30 min in darkness at laboratory temperatureBlockage with 100 μl/well 1.8NH₂SO₄

Reading of the OD at 492 nm

5.2. Indirect ELISA Test with an Anti-Salmonella Fab′Conjugate/Tetrabiotinylated or Monobiotinylated Compound

For this purpose, the antigens issuing from the culture of the threestrains of Salmonella were coated on Polysorp microplates. The followingsteps were then carried out:

-   -   Passivation for 1 h in the oven at 37° C. with 1 g/l BSA in PBS,    -   3 washings with PBS tween, then    -   Incubation of the conjugates diluted twofold in PBS for 30 min        at 37° C., 3 washings with PBS tween.

The same procedure was then followed as in the direct ELISA test fromthe deposition of streptavidin.

The indirect ELISA serves both to test the “coupling level” and thebiological reaction between the conjugate and the antigen or theantibody.

5.3 Results

The results are shown in FIGS. 2 to 8 as follows:

FIG. 2 is the graphic representation giving the OD results of the directELISA test of the anti-Listeria monocytogenes (LMO) Fab′ conjugate thatis monobiotinylated (by biotin BMCC) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of themicroplate, according to its concentration,

FIG. 3 is the graphic representation giving the OD results of the directELISA test of the anti-Salmonella Fab′ conjugate that ismonobiotinylated (by biotin BMCC) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of themicroplate, according to their concentration, and of the indirect ELISAtest of these same conjugates, but detected by means of Salmonellaantigens,

FIG. 4 is the graphic representation giving the OD results of the directELISA test of two anti-HIV Fab′ conjugates that are monobiotinylated (bybiotin BMCC) or tetrabiotinylated (by a compound according to anembodiment of the invention), fixed to the bottom of the microplate,according to their concentration,

FIG. 5 is the graphic representation giving the OD results of the directELISA test of the anti-Kallikrein Fab′ conjugate that ismonobiotinylated (by biotin BMCC) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of themicroplate, according to its concentration,

FIG. 6 is the graphic representation giving the OD results of the directELISA test of the gp160 antigen conjugate reduced with DTT that ismonobiotinylated (by biotin BMCC) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of amicroplate, according to its concentration,

FIG. 7 is the graphic representation giving the OD results of the directELISA test of surface antigen conjugates of hepatitis B that aremonobiotinylated (by biotin NHs) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of themicroplate, according to their concentration, and

FIG. 8 is the graphic representation giving the OD results of the directELISA test of the anti-Salmonella antibody conjugate that ismonobiotinylated (by biotin NHs) or tetrabiotinylated (by a compoundaccording to an embodiment of the invention), fixed to the bottom of themicroplate, according to its concentration.

The results demonstrate a significant improvement of the signal usingthe polybiotinylated molecule according to the invention, despite itshindrance. In particular, for OD of 1.8, gains can be observed such asgiven in Table 6 below:

TABLE 6 Biotin Signal BMCC Biotin NHs Tetrabiotin gain LMO 46 ng/ml NA 6ng/ml × 8 Test 1 LMO 6.0 ng/ml NA 0.4 ng/ml × 15 Test 2 HIV 0.078 μg/mlNA <0.0078 μg/ml > × 10 Antibody A OD = 1.895 HIV 0.08 μg/ml NA <0.0078μg/ml > × 10 Antibody B OD = 2.021 Kallikrein 0.287 μg/ml NA <0.0023μg/ml > × 124 OD = 2.226 Salmonella 0.012 μg/ml NA <0.0039 μg/ml > × 30Fab' direct OD = 2.825 ELISA Salmonella 0.226 μg/ml NA 0.101 μg/ml × 2.2Fab' indirect ELISA Antigen 0.01 μg/ml NA <0.0039 μg/ml > × 2.5 GP160 OD= 2.813 reduced with DTT Antigen NA 5 μg/ml OD = Traut (1/4) < × 79 HBsAd 1.320 Tetrabiotin (1/1) 0.063 μg/ml Antigen NA 2.27 μg/ml Traut (1/4)× 49 HBs Ay Tetrabiotin (1/1)0.046 μg/ml Salmonella NA (1/20) Traut(1/20) × 6.5 antibody 0.19 μg/ml Tetrabiotin (1/20) 0.029 μg/ml

EXAMPLE 6 Application to Diagnostic Tests with Strains of Listeria

The purpose of this test is to compare the Vidas signal obtained betweena tetrabiotinylated conjugate according to an embodiment and amonobiotinylated conjugate, in order to increase the sensitivity of theresponse without degrading the specificity.

6.1 Materials and Methods

Reagents Used

The tetrabiotinylated and monobiotinylated conjugates were preparedusing a Fab′ antibody fragment of Listeria monocytogenes (bioMérieux,France) and biotin BMCC and a compound as prepared in example 1,respectively, following the protocol described in section 4.3 above.

The other reagents are as follows:

LMO2 cones and well strips (bioMerieux, Reference 30 704)

PAL streptavidin (BioSPA, Ref: 045 66074)

Monobiotinylated stock solution: concentration 0.32 mg/ml of Fab′

Tetrabiotinylated stock solution: concentration 0.26 mg/ml of Fab′

LMO2 conjugate diluent (bioMerieux, Ref 500 26004)

PAL streptavidin diluent (bioMerieux, Ref 500 25992)

1 Vidas apparatus (bioMerieux)

Strains Tested

Listeria monocytogenes 4b ATCC 19115

Listeria monocytogenes 3a ATCC 51 782

Listeria monocytogenes 1/2c 83 09 024

Listeria monocytogenes 4c 83 09 031

Listeria innocua 6a 83 09 035

Listeria ivanovii 91 01 014

Listeria welshimeri 94 09 074

Methods

All the strains were cultured for 24 h at 37° C.+1° C. in LX Ref 42 120broth, then heated for 5 minutes at 95-100° C.

The strains of Listeria innocua, Listeria ivanovii and Listeriawelshimeri were used pure. These strains serve to test the specificity,because they are not recognised by the coupled antibody.

The four strains of Listeria monocytogenes were tested in dilution andwere counted. These strains serve to test the sensitivity of the test.

The 2 conjugates were adjusted to the concentration of 0.36 μg/ml ofFab′.

To use the kit of the LMO2 well strip, the initial conjugate waswithdrawn from the well X5. This well was rinsed with 600 μl ofphysiological water. Then, after having removed the physiological water,400 μl of the monobiotinylated conjugate or of the tetrabiotinylatedconjugate to be tested were added. 500 μl of sample (dilution ofstrains) were deposited in well XO and the LMO2 Vidas test was started.The test lasts about 80 minutes.

Results

The results are given in Table 7 below.

TABLE 7 Stock solutions Tetrabiotinylated Monobiotinylated Strains andcount RFV* L. mono 4b ATCC 19 115 9957 9817 1.5.10⁵ L. mono 4b ATCC 19115 4350 2285 1.5.10⁴ L. mono 4b ATCC 19 115 199 233 1.5.10³ L. mono 3aATCC 51 782 8517 6958 1.6.10⁵ L. mono 3a ATCC 51 782 1612 829 1.6.10⁴ L.mono 3a ATCC 51 782 160 78 1.6.10³ L. mono 1/2c 83 09 024 9806 87792.1.10⁵ L. mono 1/2c 83 09 024 2880 1472 2.1.10⁴ L. mono 1/2c 83 09 024309 151 2.1.10³ L. mono 4c 83 09 031 2413 1427 8.4.10⁴ L. mono 4c 83 09031 143 82 8.4.10³ L. mono 4c 83 09 031 19 10 8.4.10² L innocua 6a 83 09035 7 9 L ivanovii 91 01 014 7 3 L welshimeri 94 09 074 10 6 *RelativeFluorescent Value

The results in the table above show that the use of a conjugate having atetrabiotinylated compound according to an embodiment of the inventionserves to double the signal, and hence to increase the sensitivity ofthe test. At the same time, its use does not alter the specificity,because in this case, the signals are identical.

1. Method for preparing a compound having the formula (I):

wherein X is biotin or

Y is biotin or

Z is biotin or

V is biotin or

B is NH₂ or H, AA is a trifunctional molecule derivative when B is NH₂or a bifunctional molecule derivative when B is H, AA₁ to AA₅ are eachindependently a trifunctional molecule derivative, G₀ to G₅ are eachindependently an arm comprising at least one (—CH₂—CH₂—O) unit, n₀ to n₅are each independently a whole number between 1 and 8 and T is anantiligand or a reactive group for fixation to an antiligand, saidantiligand being capable of reacting with a ligand, the methodcomprising: (i) grafting n₀ compounds having the formula W″₀-G₀-OH,where W″₀ is an amine protecting group, n₀ and G₀ being such as definedabove, to a compound having the formula (II):

where R is a prefunctionalised resin, W and W₀ are different from oneanother and represent an amine protecting group, W being different fromW″₀, and AA is such as defined above, to obtain a compound having theformula (III):

(ii) coupling a compound having a formula (IV):

where W₁ and W′₁ are amine protecting groups that are identical to ordifferent from one another and from the W₀ and W″₀ groups, but differentfrom W, and AA₁ is such as defined above, with the compound having theformula (III) to obtain a compound having the formula (V):

(iii) grafting 2n₁ compounds having the formula W″₁-G₁-OH, where W″₁ isan amine protecting group, identical to or different from one anotherand from the other protecting groups used in this method, but differentfrom W, G₁ and n₁ being such as defined above, onto the compoundobtained in step (ii) to obtain a compound having the formula (VI):

(iv) when X in the formula (I) is biotin, passing directly to step (v);if not, when X in the formula (I) is not biotin, repeating the steps(ii) and (iii) with 2^(p-1) compounds having the formula (VII):

and 2 ^(p)n_(p) times the compound having the formula W″_(p)-G_(p)-OH,where p is a whole number between 2 and 5, and W_(p), W′_(p), W″_(p) areamine protecting groups that are identical to or different from oneanother and from the other protecting groups used in this method, butdifferent from W, and AA_(p) is a trifunctional molecule, according tothe following sequence: 1 time when Y is biotin, p then being equal to 22 times when Y is not biotin and Z is biotin, p then being equal to 2then to 3, 3 times when Y and Z are not biotin and V is biotin, p thenbeing equal to 2, 3 then 4, and 4 times when Y, Z and V are not biotin,p then being equal to 2, 3, 4 then 5, (v) deprotecting the compound thusobtained at the W″₁ or W″_(p) group, p being between 2 and 5, andcoupling with biotin, (vi) deprotecting, at the W group, the compoundthus polybiotinylated and coupling with an antiligand or a reactivegroup for fixation to an antiligand (T) and (vii) cutting the compoundthus obtained from the resin (R) to obtain a compound having the formula(I).
 2. Method according to claim 1, wherein the substituents AA₁ andAA₂, if applicable AA₃, AA₄ and AA₅, are identical and are preferably alysine derivative.
 3. Method according to claim 1, wherein thesubstituents G₀, G₁, if applicable G₂, G₃, G₄ and G₅ are identical andpreferably have the formula (—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—).
 4. Methodaccording to claim 1, wherein the whole numbers n₀, n₁, if applicablen₂, n₃, n₄ and n₅, are identical and are preferably equal to 2 or
 3. 5.Method according to claim 1, wherein the W group is methoxytrityl. 6.Method according to claim 1, wherein W₁ and W′₁ are identical and, ifapplicable, W₂ and W′₂ are identical, W₃ and W′₃ are identical, W₄ andW′₄ are identical and W₅ and W′₅ are identical.
 7. Method according toclaim 6, wherein the amine protecting groups W₁, W′₁ and, if applicable,W₂, W′₂, W₃, W′₃, W₄ and W′₄, W₅ and W′₅ are fluorenylmethoxycarbonylgroups.
 8. Method according to claim 1, wherein the substituent AA is alysine derivative and B is NH₂.
 9. Method according to claim 1, whereinX is

and Y is biotin.
 10. Compound having the formula (I):

where X is biotin or

Y is biotin or

Z is biotin or

V is biotin or

B is NH₂ or H, AA is a trifunctional molecule derivative when B is NH₂or a bifunctional molecule derivative when B is H, AA₁ to AA₄ are eachindependently a trifunctional molecule derivative, G₀ to G₅ are eachindependently an arm comprising a (—CH₂—CH₂—O) unit, n₀ to n₅ are eachindependently a whole number between 1 and 8 and T is a maleimide group,a carboxylic acid group or an antiligand.
 11. Compound having theformula (I) according to claim 10, wherein the substituents AA₁ and AA₂,if applicable AA₃, AA₄ and AA₅, are identical and are preferably alysine derivative.
 12. Compound having the formula (I) according toclaim 10, wherein the substituents G₀, G₁, if applicable G₂, G₃, G₄ andG₅, are identical and preferably have the formula(—NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO—).
 13. Compound having the formula (I)according to claim 10, wherein the whole numbers n₀, n₁, if applicablen₂, n₃, n₄ and n₅, are identical and are preferably equal to 2 or
 3. 14.Compound having the formula (I) according to claim 10, wherein X is

and Y is biotin.
 15. Compound having the formula (I) according to claim10, wherein AA is a lysine derivative and B is NH₂.
 16. Compound havingthe formula (I) according to claim 10, wherein the antiligand is a Fab′fragment.
 17. Compound having the formula (I) according to claim 10,wherein: X is

Y is biotin, AA, AA₁ and AA₂ are a lysine derivative, G₀, G₁ and G₂ havethe formula —(NH—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CO)—, n₀, n₁ and n₂ are equalto 2, B is NH₂ and T is an antiligand, preferably a Fab′ fragment. 18.Use of the compounds obtained by the method as described in claim 1 forsignal amplification in a diagnostic test.
 19. Use of the compounds asdescribed in claim 10 for signal amplification in a diagnostic test.